The centromere is that region of a eukaryotic chromosome which is attached to the spindle during nuclear division. Aside from its normal role in cell division, the centromere is also of interest since in certain autoimmune diseases anticentromere autoantibodies (ACA) are produced. This is especially true for rheumatic diseases. ACA were discovered in 1980, when it was found that certain patients with the calcinosis/Raynauds phenomenon/esophegeal dysmotility/scleroactyly/elangiectasiae (CREST) variant of scleroderma produce autoantibodies that recognize the centromere region of chromosomes. Though ACA are closely associated with the CREST syndrome, the only clinical finding common to all ACA.sup.+ individuals is Raynauds phenomenon.
Rheumatic diseases are characterized by the production of autoantibodies directed against nuclear and cytoplasmic autoantigens. The reasons for autoantibody expression are generally unknown and many theories seeking to explain the phenomenon are currently under consideration. In particular, it is not known if the autoimmune response results from a classical antigen-driven immunization or is a result of aberrations in the mechanisms that normally control the immune system. Although much progress has been made in identifying and characterizing autoantigens, the origin of antinuclear autoantibodies remains obscure. It has been suggested that autoantibodies might arise as a result of fortuitous cross-reactions exhibited by normal antibodies, as a result of chance mutations causing normal B-cell clones elicited by foreign antigen to change specificity and recognize self components, or as antiidiotypes elicited during an immune response against a viral protein. These models could explain an autoimmune response against any single epitope, but they cannot account for the ACA response, since multiple structurally independent epitopes requiring multiple chance events are targeted in virtually every affected individual.
Prior immunoblotting analysis has revealed that approximately 96% of a test group of 39 ACA.sup.+ sera recognized three chromosomal polypeptides of 17 kDA (CENP-A), 80 kDa (CENP-B), and 140 kDa (CENP-C) (Earnshaw, et al.,Journal of Clinical Investigation, 77: 426, 1986). Affinity purified antibodies to CENP-B cross-reacted with CENPs A and C, indicating that these antigens are structurally related (Earnshaw, et al., Chromosoma, 91: 313, 1985). Antibodies to CENP-B are present at high titer in all ACA.sup.+ sera examined, whereas the titer of autoantibodies to CENPs A and C is occasionally lower.
Since the various types of autoimmune disease can be characterized based upon the specificity of autoantibodies which are produced, the ability to detect autoantibodies reactive with the centromere polypeptides would be of great value in the characterization of autoimmune disease. Unfortunately, the existing commercial systems detect anticentromere autoantibodies by utilizing dividing cells which contain nuclear autoantigens other than the centromere. These systems will detect antibodies which react with nuclear polypeptides other than the centromere and, as a result, may give false positive results which can lead to an incorrect classification. In addition, these existing systems are awkward to perform and difficult to scale up to enable testing of large numbers of samples. Thus, there is considerable need for a system which efficiently detects only antibodies to the centromere and not other nuclear polypeptides.
Presently, chromosomes are sorted by reacting them with dyes which bind to DNA. The presence of these dyes is then detected by irradiating the chromosomes. Unfortunately, this irradiation can damage or break the DNA of the chromosomes thereby complicating the sorting process. Consequently, there is a need for a way to sort or classify chromosomes with minimal damage to the chromosome.